Process for producing a recombinant protein in a mycobacterium host plasmid pJAM2

ABSTRACT

This invention relates to a novel mycobacterial protein named DES, which appears to share significant amino acid sequence homology with soluble stearoyl-ACP desaturases. The results of allelic exchange experiments, indicate that the des gene may be essential to the survival of mycobacteria. These results coupled with the surface localization, the unique structure of DES, and the fact this antigen is expressed in vivo, and DES protein induces a humoral response in human patients, indicate that the DES protein provides a new target for the design of anti-mycobacterial drugs. This invention provides methods of screening molecules that can inhibit the DES enzyme activity of purified DES protein, in order to identify antibiotic molecules that are capable of inhibiting the growth or survival of mycobacteria. These methods may be practiced by using recombinant DES protein obtained from a recombinant mycobacterium host cell that was transformed with a vector containing the des gene, whose expression is controlled by regulatory or promoter sequences that function in mycobacteria. Another aspect of this invention relates to the molecules that have been identified according to the screening methods as having antibiotic activity: against mycobacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 10/914,165 filed Aug. 10,2004 (Allowed), now U.S. Pat. No. 7,067,136 which is a division ofapplication Ser. No. 10/383,675 filed Mar. 10, 2003 (Abandoned), whichis a division of application Ser. No. 09/429,370, filed Oct. 28, 1999(now U.S. Pat. No. 6,573,064), which claims the benefit under 35 U.S.C.§ 119(e) of U.S. Provisional Application No. 60/111,813, filed Dec. 11,1998, and U.S. Provisional Application No. 60/113,675, filed Nov. 4,1998, and U.S. Provisional Application No. 60/198,229, filed Oct.28,1998, the benefit of each of which is hereby claimed.

BACKGROUND OF THE INVENTION

Tuberculosis and leprosy, caused by the bacilli from the Mycobacteriumtuberculosis complex and M. leprae, respectively, are the two majormycobacterial diseases. Other mycobacteriosis caused by a typicalmycobacteria such as M. avium, M. xenopi, and M. Kansasii also representmajor health problems worldwide.

M. avium is a predominant strain isolated from T.B. patients with AIDS(Horburgh et al., 1991) and M. xenopi along with M. kansasii and M.avium, is the main agent of pulmonary infections due to opportunistmycobacteria in HIV seronegative patients. (M. Picardeau et al., 1995).

In addition, these atypical mycobacteriosis are often difficult to curebecause of the lack of efficient drugs specifically directed againstatypical mycobacteria. Pathogenic mycobacteria have the ability tosurvive within host phagocytic cells. The pathology of the tuberculosisinfection derives from the interactions between the host and thebacteria, resulting from the damage the host immune response causes ontissues (Andersen & Brennan, 1994). In addition, the protection of thehost against mycobacteria infection also depends on interactions betweenthe host and mycobacteria.

Identification of the bacterial antigens involved in these interactionswith the immune system is essential for the understanding of thepathogenic mechanisms of mycobacteria and the host immunologicalresponse in relation to the evolution of the disease. It is also ofgreat importance for the improvement of the strategies for mycobacterialdisease control through vaccination and immunodiagnosis.

Through the years, various strategies have been followed for identifyingmycobacterial antigens. Biochemical tools for fractionating andanalyzing bacterial proteins permitted the isolation of antigenicproteins selected on their capacity to elicit B- or T-cell responses(Romain et al., 1993; Sorensen et al., 1995). The recent development ofmolecular genetic methods for mycobacteria (Jacobs et al., 1991; Snapperet al., 1990; Hatful, 1993; Young et al., 1985) allowed the constructionof DNA expression libraries of both M. tuberculosis and M. leprae in theλgt11 vector and their expression in E. coli. The screening of theserecombinant libraries using murine polyclonal or monoclonal antibodiesand patient sera led to the identification of numerous antigens(Braibant et al., 1994; Hermans et al., 1995; Thole & van der Zee,1990). However, most of them turned out to belong to the group of highlyconserved heat shock proteins (Thole & van der Zee, 1990; Young et al.,1990).

The observation in animal models that specific protection againsttuberculosis was conferred only by administration of live BCG vaccine,suggested that mycobacterial secreted proteins might play a major rolein inducing protective immunity. These proteins were shown to inducecell-mediated immune responses and protective immunity in a guinea pigor a mouse model of tuberculosis (Pal & Horwitz, 1992; Andersen, 1994;Haslov et al., 1995). Recently, a genetic methodology for theidentification of exported proteins based on PhoA gene fusions wasadapted to mycobacteria by (Lim et al., 1995). It permitted theisolation of M. tuberculosis DNA fragments encoding exported proteins,including the already known 19 kDa lipoprotein (Lee et al., 1992) andthe ERP protein similar to the M. leprae 28 kDa antigen (Berthet et al.,1995).

SUMMARY OF THE INVENTION

We have characterized a new M. tuberculosis exported protein identifiedby using the PhoA gene fusion methodology. The des gene, which seemsconserved among mycobacterial species, encodes an antigenic proteinhighly recognized by human sera from both tuberculosis and leprosypatients but not by sera from tuberculous cattle. The results of allelicexchange experiments described in this application, indicate that thedes gene is essential to the survival of mycobacteria.

The amino acid sequence of the DES protein contains two sets of motifsthat are characteristic of the active sites of enzymes from the class IIdiiron-oxo protein family. Among this family, the DES protein presentssignificant homologies to soluble stearoyl-acyl carrier protein (ACP)desaturases. Three dimensional modeling demonstrates that the DESprotein and the plant stearoyl-ACP desaturase share a conserved activesite.

This invention also provides methods of identifying molecules capable ofinhibiting the growth and/or survival of Mycobacteria species. Inparticular, the methods of this invention include screening moleculesthat can inhibit the activity of the DES protein. These methods comprisethe steps of:

-   -   a) contacting the molecule with a strain of mycobacteria species        containing an active DES protein or a DES like protein or a        vector carrying an active DES protein gene or a vector        containing a polynucleotide sequence encoding the active site of        the DES protein;    -   b) measuring the inhibition of the growth of said mycobacteria        strain; and    -   c) identifying the molecule that is reacting with the DES        protein or with the active site of said protein carrying        conserved residues.

To practice the methods of this invention, the purified DES protein maybe a recombinant desaturase protein. The recombinant DES protein can beobtained from a recombinant mycobacterium host cell that was transformedwith an expression vector containing a polynucleotide encoding the DESprotein whose expression is it controlled by regulatory sequences thatfunction in mycobacteria. In one method of the invention, therecombinant expression vector is a plasmid derived from the pJAM2plasmid (e.g. pJAM21). The invention also encompasses the pJAM2 andpJAM21 plasmids, as well as recombinant host cells transformed with thepJAM2 and pJAM21 plasmids. A recombinant host cell transformed withpJAM21 has been deposited at Collection Nationale de Cultures deMicro-organisms (CNCM) in Paris, France, on Jun. 23, 1998, underaccession number I-2042.

Another aspect of this invention relates to molecules that have beenscreened according to the methods of this invention and identified ashaving antibiotic activity against mycobacteria.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with description, serve to explain the principlesof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction map of the 4.5 kb EcoRV fragment encoding the M.tuberculosis des gene.

FIG. 2A is a vector map for the pJAM2 plasmid.

FIG. 2B is the nucleotide sequence of the multi-cloning site andsurrounding regions of pJAM2. The Shine-Delgarno sequence (S.D.) isshown in bold type.

FIG. 3 shows a comparative sequence analysis of class II diiron-oxoproteins and the M. tuberculosis DES protein. Shaded residues indicatecluster ligands and probable iron ligands in the M. tuberculosis DESprotein. Bold unshaded framed letters are probable residues involved inthe network of hydrogen bonds to the cluster. Other bold lettersindicate conserved residues that are believed to participate in theO₂-binding site. Gaps introduced into the sequence of DES are indicatedby dots. Accession numbers are as follows.: V015555, Epstein-Barr virusribonucleotide reductase; M58499, Methylococcus capsulatus methanemono-oxygenase hydroxylase; M60276, Pseudomonas sp. strain CF 600 phenolhydroxylase dmpN polypeptide; M59857, Ricinus communis stearoyl-ACPdesaturase; and D38753, O. sativa stearoyl-ACP desaturase.

FIG. 4 is a Southern blot analysis of the distribution of the des genein other mycobacterial species. DNA from various mycobacterial strainswere PstI-digested, electrophoresed, transferred onto a nylon membraneby Southern blotting, and hybridized using probe B, which is shown inFIG. 1.

FIG. 5 shows an SDS-PAGE gel of soluble and insoluble extracts from E.coli expressing the DES protein on plasmid pETdes (I-1718).

FIG. 6 shows the results of ELISAs of the sensitivity of the antibodyresponse to the DES antigen of human tuberculous and non-tuberculouspatients.

FIG. 7 shows the nucleotide and derived amino acid sequences of theMycobacterium tuberculosis des gene. The underlined sequences correspondto the −35 and −10 boxes of the promoter and a Shine Delgarno sequencethat corresponds to the putative ribosomal attachment site,respectively. The adenosine labeled “+1” corresponds to thetranscription initiation site.

FIG. 8 is a table of the bacterial strains and plasmids used in thisapplication.

FIG. 9 is a Western blot showing the recognition of the purified DESprotein by antibodies from M. bovis and M. tuberculosis-infected humansand cattle.

FIG. 10 shows the inducible expression of the gene encoding the M.leprae 35 kDa protein in M. smegmatis in the presence or absence of theacetamidase inducer acetamide. Section (A) is an SDS-PAGE gel ofbacterial sonicates and purified protein. Section (B) is a Western blotof a corresponding gel analyzing reactivity with the anti-M. leprae mAbCS38. Lane 1 corresponds to M. smegmatis harboring pJAM4 grown in theabsence of acetamide; lane 2 corresponds to M. smegmatis harboring pJAM4grown in the presence of acetamide; lane 3 corresponds to purified M.leprae 35 kDa protein.

FIG. 11 is actable representing the quantification of the M. lepraeprotein produced in recombinant M. smegmatis in the presence or absenceof the acetamidase inducer acetamide. Results are expressed as the meanvalue±SEM of three experiments. Suc: is an abbreviation for succinate;Suc/Act: is an abbreviation succinate plus acetamide.

FIG. 12 is a graph representing the recognition of the recombinant M.leprae 35 kDa protein by lepromatous leprosy sera. In the legend, M. smg35 kDa: represents M. smegmatis-derived 35 kDa protein; M. smg 35kDa-HIS: represents M. smegmatis-derived, histidine-tagged 35 kDaprotein; and E. coli 35 kDa: represents E. coli-derived 35 kDa protein.

FIG. 13 is a Western blot showing induction of the gene encoding the M.tuberculosis DES antigen in M. smegmatis using the pJAM2 expressionsystem. Ten μg of cell sonicate from bacteria grown in the absence (−)or presence (+) of acetamide were added to each lane and the transferredgel was immunoblotted with anti-DES murine polyclonal antibody. WTrepresents wild-type M. smegmatis mc²155; and MYC1553 represents M.smegmatis harboring pJAM21. Sonicates from two transformants are shown.The location of the DES antigen is indicated.

DETAILED DESCRIPTION

Using the Pho A gene fusion methodology, we identified a new 37 kDaMycobacterium tuberculosis protein, designated DES. This 37 kDa exportedprotein contains conserved amino acid residues which are characteristicof class II diiron-oxoproteins. Proteins from that family are allenzymes that require iron for activity. They include ribonucleotidereductases, hydrocarbon hydroxylases and stearoyl-ACP desaturases. TheM. tuberculosis DES protein only presents significant homologies toplant stearoyl-ACP desaturases (44% identity at the nucleotide level,and 30% identity at the amino-acid level), which are exported enzymes asthey are translocated across the chloroplastic membranes (Keegstra &Olsten, 1989).

Three-dimensional modeling of the DES protein based on homology with theRicinus communis Δ9 stearoyl-ACP indicates that the DES protein sharessignificant structural features with the plant stearoyl-ACP desaturases.Most importantly, the active site of the DES protein and the plant Δ9stearoyl-ACP desaturase are conserved, suggesting that DES isevolutionarily related to the plant desaturases.

The plant stearoyl-ACP desaturase can be used for the screening and theselection of new compounds inhibiting the activity of the enzyme andconsequently then tested for the modulation of the properties of DESprotein in vivo in a mycobacterial strain, such as M. tuberculosis or invitro on a purified DES protein. This result suggests that the DESprotein could be involved in the mycobacterial fatty acid biosynthesis.

Furthermore, the localization of the protein outside the cytoplasm wouldbe consistent with its role in the lipid metabolism, since lipidsrepresent 60% of the cell wall constituents and that part of thebiosynthesis of the voluminous mycolic acids containing 60 to 90 carbonatoms occurs outside the cytoplasm. Among all the different steps of thelipid metabolism, desaturation reactions are of special interest, firstbecause they very often take place at early steps of lipid biosynthesisand secondly because, through the control they have on the unsaturationrate of membranes, they contribute to the adaptation of mycobacteria totheir environment (Wheeler & Ratledge, 1994). An enzyme system involvinga stearoyl-Coenzyme A desaturase (analog of the plantstearoyl-ACP-desaturases), catalyzing oxydative desaturation of the CoAderivatives of stearic and palmitic acid to the corresponding Δ9monounsaturated fatty acids has been biochemically characterized inMycobacterium phlei (Fulco & Bloch, 1962; Fulco & Bloch, 1964;Kashiwabara et al., 1975; Kashiwabara & Sato, 1973). This system wasshown to be firmly bound to a membranous structure (Fulco & Bloch,1964). Thus, M. tuberculosis stearoyl-Coenzyme A desaturase (Δ9desaturase) is expected to be an exported protein.

Sonicated extracts of E. coli expressing the DES protein were assayedfor Δ9 desaturating activity according to the method described by(Legrand and Bensadoun, 1991), using (stearoyl-CoA) ¹⁴C as a substrate.However, no Δ9 desaturating activity could be detected. This result isprobably linked to the fact that desaturation systems are multi-enzymecomplexes involving electron transport chains and numerous cofactors,often difficult to render functional in vitro. Since E. coli andmycobacteria are very different from a lipid metabolism point of view,in E. coli, the M. tuberculosis recombinant Δ9 desaturase might notdispose of all the cofactors and associated enzymes required foractivity or might not interact properly with them. Moreover, not allcofactors involved in the Δ9 desaturation process of mycobacteria areknown, and they might be missing in the incubation medium.

However, if the DES protein encodes a Δ9 desaturase, an interestingpoint concerns its primary sequence. Indeed, all animal, fungal, and theonly two bacterial Δ9 desaturases sequenced to date (Sakamoto et al.,1994) are integral membrane proteins which have been classified into athird class of diiron-oxo proteins on the basis of their primarysequences involving conserved histidine residues (Shanklin et al.,1994). The plant soluble Δ9 desaturases are the only desaturases topossess the type of primary sequence of class II diiron-oxo proteins(Shanklin & Somerville, 1991). No bacteria have yet been found whichhave a plant type Δ9 desaturase.

As shown by immunoblotting and ELISA experiments, the DES protein is ahighly immunogenic antigen which elicits a B-cell response in 100% ofthe tuberculosis M. bovis or M. tuberculosis-infected human patientstested, independently of the form of the disease (extrapulmonary orpulmonary). It also elicits an antibody response in lepromatous leprosypatients. Interestingly, although more sera would need to betested,tuberculous cattle do not seem to recognize the DES antigen.Furthermore, the ELISA experiments showed that it is possible todistinguish tuberculosis patients from patients suffering from otherpathologies on the basis of the sensitivity of their antibody responseto the DES antigen. The DES antigen is therefore a good candidate to beused for serodiagnosis of tuberculosis in human patients.Non-tuberculous patients may recognize the DES protein at a low levelbecause they are all, BCG-vaccinated individuals (BCG expressing theprotein), or because of cross-reactivity of their antibody response withother bacterial antigens. It would now be interesting to know whetherthe DES antigen possesses in addition to its B-cell epitopes, T-cellepitopes, which are the only protective epitopes in the hostimmunological response against pathogenic mycobacteria. If the DESprotein is also a good stimulator of the T-cell response in a majorityof tuberculosis patients, it could be used either individually or aspart of a cocktail of antigens in the design of a subunit vaccineagainst tuberculosis.

To gain insights into the precise function of this atypical bacterialenzyme, we attempted to interrupt the des gene in the vaccine strain M.bovis BCG by allelic exchange. In a first experiment, no allelicexchange mutants were obtained, suggesting that the des gene isessential to the viability of mycobacteria. To investigate thishypothesis, the first experiment was repeated using a M. bovis BCGstrain transformed with a second wild-type copy of the des gene. Usingthis transformed M. bovis BCG strain, we obtained allelic exchangemutants, in which a wild-type copy of the des gene was replaced by aninactivated copy of the des gene. Thus, allelic exchange was onlypossible if a second copy of the wild-type des gene had been insertedinto the M. bovis BCG chromosome. This result strongly suggests that desis an essential gene in mycobacteria from the M. tuberculosis complex.

Coupled with the localization of DES at the surface of the tuberclebacilli, and its structural originality (this enzyme's structure differsfrom all the mammalian and bacterial desaturase structures identified todate), the results of these experiments suggest that the DES proteincould be a target for designing new anti-mycobacterial drugs.

Fundamental to the analysis of the biological function and immunologicalrelevance of mycobacterial proteins is their production in a recombinantform that resembles that of their native counterpart. Recent studiesanalyzing both structure (Garbe et al., 1993; Triccas et al., 1996) andimmunogenicity (Garbe et al., 1993; Roche et al., 1996; Triccas et al.,1996) of recombinant proteins obtained from fast growing mycobacterialhosts, such as Mycobacterium smegmatis, have demonstrated superiorityover the same protein purified from E. coli expression systems. Althoughsuch approaches for the production of recombinant mycobacterial proteinsappear advantageous, two major obstacles lie in the way of furtherimprovement to these systems. The first is the inability to regulatehigh-level expression of foreign genes in M. smegmatis, analogous tosystems such as induction of the lac promoter in E. coli (de Boer etal., 1983). Secondly, no simple, efficient and widely adaptable methodfor the purification of proteins from recombinant mycobacteria has beendescribed.

In this application, we attempt to resolve these two problems. First, wedescribe the construction of a vector, pJAM2, that utilizes the promoterof the inducible acetamidase enzyme of M. smegmatis to drive high-levelexpression of foreign genesin M. smegmatis. The 47 kDa acetamidaseenzyme of M. smegmatis NCTC 8159 permits the growth of the organism onsimple amides as the sole carbon source and is highly inducible in thepresence of acetamide (Mahenthiralingam et al., 1993). This property hasbeen previously used to assess luciferase as a reporter of geneexpression in mycobacteria (Gordon et al., 1994) and to develop amycobacterial-conditional antisense mutagenesis system (Parish et al.,1997b). In this study, we constructed a vector that allows for regulatedhigh-level expression of foreign genes in mycobacteria by virtue of theM. smegmatis acetamidase promoter.

Recombinant M. leprae 35 kDa antigen produced in this system representedapproximately 8.6% of the total M. smegmatis soluble protein, with theamount of protein produced greater than that when the same gene isplaced under the control of the strong mutated β-lactamase promoter ofM. fortuitum (FIG. 3).

Secondly, we demonstrate the simple and efficient purification of theencoded antigens by use of a poly-histidine tag and one step Ni⁺⁺affinity chromatography. The addition of the histidine tag did notappear to affect the conformation or immunogenicity of the recombinantprotein, suggesting the system described may be extremely useful for thepurification of structurally and immunologically intact recombinantmycobacterial proteins from fast-growing mycobacterial hosts.

The ability to produce recombinant products in a form that closelyresembles their native state is important in the study of microbialantigens and enzymes. Recent studies have highlighted the superiority ofrecombinant protein purified from mycobacterial hosts compared to E.coli-derived products, as assessed by structural and immunologicalanalysis (Garbe et al., 1993; Roche et al., 1996; Triccas et al., 1996).Previously we have demonstrated that sera from leprosy patients wouldonly recognize the M. leprae 35 kDa. protein if the antigen was producedin a form that resembles the native protein, based on the binding ofconformational dependent mAbs and FPLC size exclusion analysis (Triccaset al., 1996). We reconfirm such a finding with protein produced usingthe acetamidase promoter expression system (FIG. 12). Furthermore, theaddition of 6 histidine residues to the C-terminus of the recombinantprotein does not appear to affect its conformation, as there is littledifference in the recognition of leprosy sera by histidine-tagged andnonhistidine-tagged 35 kDa protein (FIG. 12). The efficient expressionof the 6-histidine tag in mycobacteria and the simple and effectivepurification of our model protein by Ni-NTA affinity chromatography(FIG. 10) suggest that this versatile purification system, usedsuccessfully in a number of eucaryotic and procaryotic expressionsystems (Crowe et al., 1994), could be more widely applied tomycobacterial proteins. Furthermore, the histidine purification systemovercomes the problems involved with antibody affinity chromatographyused in a number of studies to purify recombinant mycobacterial proteins(Roche et al., 1996; Triccas et al., 1996), such as the unavailabilityof appropriate antibodies or the presence of homologues capable ofbinding the antibody. Together, these results suggest an application forthe pJAM2 expression vector in the production of native-like recombinantmycobacterial proteins that can be exploited to correctly analyzeprotein function and antigenicity.

The invention will be further clarified by the following examples, whichare intended to be purely exemplary of the invention.

EXAMPLES

Bacteria, Media and Growth Conditions

The bacterial strains and plasmids used in this study are listed in FIG.8. E. coli DH5a or BL21 (DE3) pLysS cultures were routinely grown inLuria B medium (Difco) at 37° C. Mycobacterium cultures were grown inMiddlebrook 7H9 medium (Difco) supplemented with Tween 0.05%, glycerol(0.2%) and ADC (glucose, 0.2%; BSA fraction V, 0.5%; and NaCl, 0.085%)at 37° C. When required, antibiotics were added at the followingconcentrations: ampicillin (100 μg/ml)., kanamycin (20 μg/ml).

Human and Cattle Sera

Serum specimens from 20 individuals with pulmonary or extra-pulmonarytuberculosis (M. tuberculosis infected) were obtained from the Blignysanatorium (France). Six sera from M. bovis infected human tuberculouspatients and 24 sera from BCG-vaccinated patients suffering from otherpathologies were respectively obtained from Institut Pasteur,(Madagascar), and the Centre de Biologie Médicale spécialisée (CBMS)(Institut Pasteur, Paris). Sera from tuberculous cattle (M. bovisinfected) were obtained from CNEVA, (Maison Alfort).

Subcloning Procedures

Restriction enzymes and T4 DNA ligase were purchased from Gibco/BRL,Boehringer Mannheim and New England Biolabs. All enzymes were used inaccordance with the manufacturer's recommendations. A 1-kb ladder of DNAmolecular mass markers was from Gibco/BRL. DNA fragments used in thecloning procedures were gel purified using the Geneclean II kit (BIO 101Inc., La Jolla, Calif.). Cosmids and plasmids were isolated by alkalinelysis (Sambrook et al., 1989). Bacterial strains were transformed byelectroporation using the Gene Pulser unit (Bio-Rad Laboratories,Richmond, Calif.).

Southern Blot Analysis and Colony Hybridization

DNA-fragments for radiolabeling were separated on 0.7% agarose gels(Gibco BRL) in a Tris-borate-EDTA buffer system (Sambrook et al., 1989)and isolated from the gel by using Geneclean II (BIO 101). Radiolabelingwas carried out with the random primed labeling kit Megaprime (Amersham)with 5 μCi of (α⁻³²P)dCTP, and unincorporated label was removed bypassing through a Nick Column (Pharmacia). Southern blotting was carriedout in 0.4 M NaOH with nylon membranes (Hybond-N+, Amersham) accordingto the Southern technique (Southern, 1975), prehybridization andhybridization was carried out as recommended by the manufacturer usingRHB buffer (Amersham). Washing at 65° C. was as follows: two washes with2×SSPE (150 mM NaCl, 8.8 mM NaH₂PO₄, 1 mM EDTA pH 7.4) -SDS 0.1% of 15minutes each, one wash with 1×SSPE-SES 0.1% for 10 minutes, two washeswith 0.7×SSPE-SDS 0.1% of 15 minutes each. Autoradiographs were preparedby exposure with X-ray film (Kodak X-OMAT) at −80° C. overnight. Colonyhybridization was carried out using nylon membrane disc (Hybond-N+ 0.45μm, Amersham). E. coli colonies adsorbed on the membranes were lysed ina (0.5M NaOH, 1.5M NaCl) solution, before being placed for one minute ina microwave oven to fix the DNA. Hybridization and washes were describedfor the Southern blotting analysis.

DNA Sequencing and Analysis

Sequences of double-stranded plasmid DNA were determined by thedideoxy-chain termination method (Sanger et al., 1977) using the TaqDye. Deoxy Terminator Cycle sequencing Kit (Applied Biosystems), on aGeneAmp PCR System 9600 (Perkin Elmer), and run on a DNA AnalysisSystem-Model 373 stretch (Applied Biosystems). The sequence wasassembled and processed using DNA strider (CEA, France) and theUniversity of Wisconsin Genetics Computer Group *UWGCG) packages. TheBLAST algorithm (Altschul et al., 1990) was used to search protein databases for similarity.

Expression and Purification of the DES Protein in E. coli

A 1043 bp NdeI-BamHI fragment of the des gene was amplified by PCR usingnucleotides JD8:

(5′-CGGCATATGTCAGCCAAGCTGACCGACCTGCAG-3′) (SEQ ID. NO:1), and JD9:

(5′CCGGGATCCCGCGCTCGCCGCTCTGCATCGTCG-3′)(SEQ ID NO:2), and cloned intothe NdeI-BamHI sites of pET14b (Novagen) to generate pET-des. PCRamplifications were carried out in a DNA thermal Cycler (Perkin Elmer),using Taq polymerase (Cetus) according to the manufacturer'srecommendations. PCR consisted of one cycle of denaturation (95° C., 6min) followed by 25 cycles of amplification consisting of denaturation(95° C., 1 min), annealing (57° C., 1 min), and primer extension (72°C., 1 min). In the pET-des vector, the expression of the des gene isunder control of the T7 bacteriophage promoter and the DES antigen isexpressed as a fusion protein containing six histidine residues.Expression of the des gene was induced by addition of 0.4 mM IPTG in theculture medium. The DES protein was purified by using a nickel-chelateaffinity resin according to the recommendations of the supplier (Qiagen,Chatsworth, Calif.)SDS-PAGE and Immunoblotting

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) wascarried out as described by (Laemmli, 1970). For Western blottingexperiments (immunoblotting), approximately 10 μg of DES purifiedprotein were run on a SDS-polyacrylamide gel and transferred onnitrocellulose membranes (Hybond C extra, Amersham) using a Bio-Rad minitransblot apparatus according to the recommendations of the manufacturer(Bio-Rad Laboratories, Richmond, Calif). Transfer yield was visualizedby transient staining with Ponceau Rouge. The membrane were incubatedwith human patient or cattle sera diluted 1/200 at 37° C. for 1 hour andwith a goat anti-human (Promega) or rabbit anti-cattle (Biosys) IgGalkaline phosphatase-conjugated secondary antibody diluted 1/2500′ for30 minutes at 37° C. The color reaction was performed by addition of5-bromo-4-chloro-3-indolylphosphate (0.165 mg/ml) and toluidinumnitroblue tetrazolium (0.33 mg/ml) as substrates.

ELISA

The human or cattle sera were tested for antibodies against DES byenzyme-linked immunosorbent assay (ELISA). The 96-well micro-titer trays(Nunc, Rochester, N.Y.) were coated with 0.1 μg (per well) of purifiedDES protein in guanidine hydrochloride buffer A (6 M guanidinehydrochloride, 0.1 M NaH₂PO₄, 0.01 M Tris, pH 8) (1 h at 37° C. and 16 hat 4° C). After three washes, wells were saturated with bovine serumalbumin 3% in phosphate buffered saline (PBS) for 30 min. at roomtemperature. After three washes, sera diluted from 1/500° to 1/3200° inbuffer (PBS, 0.1% Tween 20, 1% bovine serum albumin) were added to thewells for 2 h at 37° C. After three washes, the wells were treated withgoat anti-human IgG-alkaline phosphatase conjugate (Promega, Madison,Wis.) diluted 1/4000° for 1 h at 37° C. Then, 4 mg ofp-nitrophenylphospate per ml were added as substrate. After 20 minutesof incubation at 37° C., the plates were read photometrically at anoptical density of 405 nm in micro-ELISA Autoreader (Dynatech, Mames laCoquette, France)

Statistics

Antibody responses of the different sera tested were compared by usingthe Student t test. P≧0.05 was considered nonsignificant.

Nucleotide Sequence and Accession Number

The nucleotide sequences of des has been deposited in the GenomeSequence Data Base (GSDB) under the accession number U49839.

Cloning of the des Gene

The construction of a fusion library of M. tuberculosis genomic DNA tothe phoA gene and its expression in M. smegmatis, described by (Lim etal., 1995), led to the isolation of several PhoA⁺ clones. pExp421 is theplasmid harbored by one of the PhoA⁺ clones selected from this library.Detection of enzymatically active alkaline phosphatase indicated thatthe pExp421 insert contains functional expression and exportationsignals. Restriction analysis showed that pExp421 carries a 1.1 kbinsert. Partial determination of its sequence identified a 577 bp ORF,named des, fused in frame to the phoA gene and presenting two motifs, of9 and 14 amino acids, conserved with soluble stearoyl-acyl-carrierprotein desaturases (Lim et al., 1995).

To isolate the full-length des gene, the M. tuberculosis H37Rv pYUB18genomic cosmid library (Jacobs et al., 1991), was screened by colonyhybridization with the 1.1 kb probe (probe A, see FIG. 1). Twohybridizing cosmids named C₃ and C₄ were selected for further isolationof the gene. C₃ and C₄ were cut with several restriction enzymes andsubjected to Southern blot analysis using the 1.1 kb fragment as aprobe.

The EcoRV restriction profile revealed a single hybridizing fragment of4.5 kb which was subcloned into pBluescript KS⁻(Stratagene, La Jolla,Calif.) to give plasmid pDS-des.

Characterization of the des Gene

The DNA sequence of the full des ORF was determined (FIG. 7). The desgene was shown to cover a 1017 bp region, encoding a 339 amino acidprotein with a calculated molecular mass of 37 kDa. The ORF starts witha potential ATG start codon at position 549, and ends with a TAG stopcodon at position 1565. There is a potential Shine-Delgarno motif(GGAGG) at position −8 upstream of the ATG. The G+C content of the ORF(62%) is consistent with the global GC content observed in themycobacterial genome. The nucleotide and deduced amino acid sequences ofthe des gene were compared to sequences in databases. They showed veryhigh homologies to the M. leprae aadX gene located on cosmid B2266,deposited in GenBank as part of the M. leprae genome sequencing project(GenBank accession number n°. U15182). Within the coding region, the DNAsequences were 79% identical while the encoded proteins were 80%identical (88% including conserved residues). The des gene also scoredsignificantly against soluble stearoyl-ACP desaturases: 44% identity atthe nucleotide level, 30% identity (51% including conserved residues) atthe amino acid level, to the Oryza saliva stearoyl-ACP desaturase(accession n°. D38753).

Although the detection of phoA enzymatic activity in the M. smegmatisclone harboring the pEXp421 suggests the DES protein is exported, nostructural similarities were found between the DES protein N terminalamino acids and signal sequences of bacterial exported proteins (Izard &Kendall, 1994).

As in the M. leprae genome, a second ORF presenting high homologies ofthe M. leprae putative NtrB gene (cosmid B2266), is located downstreamof the des gene in M. tuberculosis. Interestingly, the two ORF, des andNtrb, are separated in M. tuberculosis by two direct repeats of 66nucleotides overlapping on 9 nucleotides (FIG. 2).

The DES Protein Presents the Conserved Amino Acid Motifs of the Class IIDiiron-oxo Proteins

Further analysis of the amino acid sequence of the DES protein revealedthe presence of conserved motifs found only in class II diiron-oxoproteins (Fox et al., 1994) (FIG. 3). These proteins are oxo-bridgeddiiron clusters (Fe—O—Fe) containing proteins. They possess in theirsecondary structure 4 alpha helices involved in the protein-derivedcluster ligands. As revealed by X-ray structure studies, in theseproteins, the diiron axis is oriented parallel to the long axis of thefour helix bundle with ligands arising from four noncontiguous helices,B, C, E and F. M. tuberculosis DES protein appears to have the sameactive site residues as the class II diiron-oxo enzymes. This includesGlu and His residues (E₁₀₇ and H₁₁₀ in helix C, E₁₆₇ in helix E and E₁₉₇and H₂₀₀ in helix F) that are ligands to the iron atoms, Asp, Glu andArg residues (E₁₀₆ and R₁₀₉ in helix C, D₁₉₆ in helix F) that areinvolved in a hydrogen-bonding network to the cluster and, Ile and Thrresidues that may be part of the O₂-binding site (T₁₇₀ in helix E, I₁₉₃in helix F). Thus, the M. tuberculosis DES protein contains in itsprimary sequence a conserved EEXXH (SEQ ID NO:3) motif and a conservedDEXXH (SEQ ID NO:4) motif, where X represents any amino acid. Theconserved motifs are separated by 85 amino acids.

The class II diiron-oxo protein family contains up to dateribonucleotide reductases, hydrocarbon hydroxylases (methanemono-oxygenase, toluene-4-mono-oxygenase and phenol hydroxylase) andsoluble-ACP desaturases. On the overall sequence alignment the DESprotein presents higher homology to soluble stearoyl-ACP desaturasesthan to ribonucleotide reductases or bacterial hydroxylases. Thepercentage identity at the amino acid level of the DES protein was saidto be 30% with the Oryza sativa stearoyl-ACP desaturases, whereas it isonly 17% with the Methylococcus capsulatus methane mono-oxygenase(accession n°. M60276) and 17.7% with the Epstein Barr ribonucleotidereductase (accession n°. V01555). Homologies to the soluble Δ9desaturases mostly concern the amino acids located within the activesite in helices C, E, and F (FIG. 3).

The method according to the invention can be carried out for thescreening and selection of molecules interacting with the enzymaticactivity of DES protein, for example, for acyl-ACP desaturase normallyproduced by higher plants.

The DES Protein Shares Structural Features with the Plant acyl-ACPDesaturases

The three-dimensional structure of the DES protein was modeled based onhomology with the Ricinus communis Δ9 stearoyl-ACP desaturase. Thestructure of this plant desaturase was determined by proteincrystallography to 2.4 Å resolution (Lindqvist et al., 1996). The modelobtained has no Ramachandran outliers, has an excellent stereochemistryfor both main chain and side chains and has no bad contacts.

302 residues out of the 337 total residues of the M. tuberculosis enzymecould be built based on the template's structure and aligned with anr.m.s. of 0.34 Å for their Cα atoms. These 302 DES residues share 26%sequence identity with the residues of plant Δ9 stearoyl-ACP desaturase.Thus, the structures of these 302 residues in the model represent a goodapproximation of their true structure.

The plant Δ9 stearoyl-ACP desaturase and DES protein share almostcomplete sequence identity in the areas encoding the four helices, whichinclude the ligands for the bi-nuclear iron center, as well as in thesurrounding areas and in the area around the catalytic site. Therefore,one can be confident with the structure of the residues located withinthese areas that share substantial amino acid identity. (FIGS. 3 a and 3b) These areas include the part of the fatty acid binding site which isclose to the active site. From the structure of the Δ9 stearoyl-ACPdesaturase it was concluded that the fatty acid part of the substrate iscompletely buried in the enzyme, in a deep hydrophobic channel,positioning the site of desaturation between carbon 9 and 10 in the areaof the active site close to the binuclear iron center. (Lindqvist etal., 1996). The shape of the channel forces the substrate to bind in aconfirmation close to the product's cis-configuration. From amino acidsequence comparisons of plant desaturases it was further concluded thatthe size of the amino acid side chains at the bottom of this channeldetermines the chain length beyond the point of double bond insertionthat can be accepted by the various plant enzymes. (Cahoon et al.,1997). In the DES protein, the active site is completely conserved,suggesting that DES is evolutionarily related to the plant desaturases.If DES catalyzes a desaturation reaction, judging from the conservedshape of the substrate's pocket, the product of the enzymatic reactionwould have a cis-configuration around the introduced double bond.Inspection of the bottom of the substrate channel in the model of theDES protein shows that the exchange of threonine T181 in the plant Δ9stearoyl-ACP desaturase for the bulkier glutamine in DES (Q145) hasshortened the pocket significantly. This implies that the substrate inDES would have a maximum of seven carbons beyond the point of doublebond insertion as compared to nine carbons in the plant stearoyl-ACPdesaturase. Also, the replacement of methionine M114 in the plant enzymeby a negatively charged glutamic acid in DES (E85) could indicate thatthe substrate for the Des protein carries a polar or even positivelycharged group that can interact with this sidechain. Alternatively, thepolarity could make it difficult for hydrophobic fatty acid tails toreach the bottom of the already shorter cavity, thereby further limitingthe number of possible carbons beyond the point of doublebond insertion(e.g., to five carbons). Other amino-acid substitutions in the bindingcleft do not affect the nature, shape and size of the substrate'sbinding cavity.

The electrostatic potential surface of the Δ9 stearoyl-ACP desaturaseand of the DES protein around the entrance of the substrate's bindingchannel are very different. This difference indicates that the DESprotein and the plant Δ9 stearoyl-ACP desaturase may require differentassociated cofactors for activity and, in particular, different forms offatty acid substrates.

Distribution of the des Gene in Other Mycobacterial Species

The presence of the des gene in PstI-digested chromosomal DNA fromvarious mycobacterial strains was analyzed by Southern blotting (FIG.4). The probe used (probe B) is a PCR amplification productcorresponding to nucleotides 572 to 1589 (see FIG. 1). The probehybridized on all mycobacterial genomic DNA tested. Strong signals weredetected in M. tuberculosis, M. bovis, M. bovis BCG, M. Africanum and M.avium. Weaker signals were visible in M. microti, M. xenopi, M.fortuitum and M. smegmatis. Thus, the des gene seems to be present insingle copy at least in the slow growing M. tuberculosis, M. bovis, M.bovis BCG, M. africanuum, M. avium and M. xenopi as well as in the fastgrowing M. smegmatis.

Expression of the des Gene in E. coli

In order to over express the DES protein, the des gene was subclonedinto the bacteriophage T7 promoter-based expression vector pET14b(Novagen). A PCR amplification product of the des gene (see material andmethods) was cloned into the NdeI-BamHI sites of the vector, leading tothe plasmid pET-des. Upon IPTG induction of E. Coli BL21 DE3 pLysS cellsharboring the plasmid pET-des, a protein of about 40 kDa wasoverproduced. The 40 kDa size of the overproduced protein correspondswith the molecular mass calculated from the deduced polypeptide. Asshown in FIG. 5, the great majority of the overproduced DES protein ispresent in the insoluble matter of E. coli cells. This probably resultsfrom the precipitation of the over-concentrated protein in E. colicytoplasm resulting in the formation of inclusion bodies. To be able todissolve the protein, the purification was carried out using a nickelchelate affinity resin under denaturing conditions in guanidinehydrochloride buffers. Among all the conditions tested (pH, detergents,etc.), the only condition in which the protein could be eluted withoutprecipitating in the column and remain soluble, was in a buffercontaining 6 M guanidine hydrochloride.

Immunogenicity of the DES Protein after Infection

Twenty serum samples from M. tuberculosis infected human patients (4with extra-pulmonary tuberculosis, 15 with pulmonary tuberculosis and 1with both forms of the disease), 6 sera from M. bovis infected humanpatients and 4 sera from M. bovis infected cattle were tested eitherpooled or taken individually in immunoblot experiments to determine thefrequency of recognition of the purified DES protein by antibodies frominfected humans or cattle. 20 out of the 20 sera from the M.tuberculosis infected human patients and 6 out of the 6 sera from the M.bovis infected human patients recognized the recombinant antigen asshown by the reaction with the 37 kDa band, (FIG. 9). Furthermore, apool of sera from human lepromatous leprosy patients also reactedagainst the DES antigen.

In contrast, the pool of serum specimens from M. bovis infected cattledid not recognize the DES protein. These results indicate that the DESprotein is highly immunogenic in tuberculosis human patients. Bothpulmonary and extra-pulmonary tuberculosis patients recognize theantigen.

Magnitude of Human Patients' Antibody Responses

An enzyme-linked immunosorbent assay (ELISA) was used to compare thesensitivity of the different serum samples from 20 tuberculosis patients(15 infected by M. tuberculosis and 5 infected by M. Bovis) to the DESantigen. This technique was also carried out to compare the sensitivityof the antibody response to DES of the 20 tuberculosis patients to theantibody response of 24 patients (BCG-vaccinated) suffering from otherpathologies. As shown in FIG. 6, patients suffering from pathologiesother than tuberculosis, react at low level to the DES antigen (averageOD₄₀₅=0.17 for a serum dilution 1/100⁴) The average antibody responsefrom the tuberculosis patients infected by M. tuberculosis or M. bovisagainst the same antigen is much more sensitive (OD₄₀₅=0.32 andOD₄₀₅=0.36 respectively, for a serum dilution 1/100⁴). This differencein the sensitivity of the immunological response is statistically highlysignificant at every dilution from 1/50^(a) to 1/3200^(a) as shown by aStudent I₉₅ test (I₉₅=5.18, 6.57, 6.16, 5.79, 4.43, 2.53 and 1.95, atsera dilutions 1/50^(a), 1/100^(a), 1/200^(a), 1/400^(a), 1/800^(a),1/600^(a) and 1/3200^(a), respectively). No differences in thesensitivity of the antibody response was noticed between patientssuffering from pulmonary or extra-pulmonary tuberculosis.

Allelic Exchange of des Gene

We constructed an inactivated copy of the des gene by inserting into theXhoI site of the ApaI/SacI restriction fragment carrying the des gene(Jackson et al., 1997), a kanamycin (Km) resistance cassette. This(des:Km) construct was then inserted, along with the XylE gene, whichencodes the Pseudomonas catechol dioxygenase conferring uponmycobacteria a yellow color when sprayed with catechol (Pelicic et al.,1997), into the pJQ200 plasmid, a pBluescript-derived E. coli vectorcarrying the sacB gene. The resulting vector was called pJQdKX.

In a first experiment, we transformed M. bovis BCG with pJQdKX and triedto select mutants resulting from allelic exchange events inside the deslocus by using a two step procedure such as the one described by(Pelicic et al., 1996) In the first step, we selected, onkanamycin-containing medium, a transformant that has integrated thewhole vector inside its chromosome by a single crossing-over within thedes locus. In the second step, using the counter-selection properties ofthe sacB gene, we selected bacteria that have undergone a secondintrachromosomal crossing-over, resulting in the replacement of the wildtype copy of the des gene by its inactivated copy (des:Km), i.e.,allelic exchange mutants.

Although at the first step of the procedure, 100% of the transformantsresulted from the integration of the pJQdKX vector by a singlehomologous recombination event, no allelic exchange mutants wereobtained after the second selection step. 99.53% of the (Km, Sucrose)resistant colonies obtained at the end of the selection procedure wereXylE+, indicating that they still carried the vector in their chromosomeand probably also carried mutations in the sacB gene resulting in theirsucrose-resistant phenotype. The 0.47% XylE-remaining colonies possiblycarried mutations in both the sacB and the XylE genes since geneticanalysis (genomic hybridization, PCR) indicated they were notdes-allelic exchange mutants. This result suggests that the des genemight be essential to M. bovis BCG.

In order to investigate this hypothesis, we performed a secondexperiment in which we inserted, using an integrative vector pAV6950(Moniz-Pereira et al., 1995), a second wild type copy of the des gene(carried on a ApaI-SacI restriction fragment; see above) in thechromosome of a M. bovis BCG transformant resulting from the firstselection step described above. The resulting M. bovis BCG thuscontained two wild type copies of the des gene in addition to the(des:Km) copy carried by the inserted pJQdKX vector. When the secondselection step was applied on a culture of this bacteria, 34% of the(Km-sucrose)-resistant colonies obtained were XylE-. Genetic analysis ofthese candidates revealed that all of them corresponded to allelicexchange mutants. The other 66% (Km-sucrose)-resistant and XylE+colonies probably carried mutations in the sacB gene.

Construction of the Acetamidase Promoter Expression Vector pJAM2

The acetamidase promoter region was amplified from plasmid PAMI1, whichcontains the M. smegmatis NCTC 9449 inducible acetamidase gene andupstream region (Mahenthiralingam et al., 1993), by use of primers HIS5:

(CACGGTACCAAGCTTTCTAGCAGA) (SEQ ID NO:38), and HIS7:

(GTCAGTGGTGGTGGTGGTGGTGTCTAGAAGTACTGGATCCGAAAACTACCTCG) (SEQ ID NO:39).The resulting 1.6 kb fragment was cloned into plasmid pJEM12 (Timm etal., 1994b) to give plasmid pJAM2 (FIG. 2A). The coding region of the M.leprae 35 kDa protein was amplified by primers JN8:(TAGCTGCAGGGATCCATGACGTCGGCT) (SEQ ID NO:40), and 35REV2(GTGTCTAGACTTGTACTCATG) (SEQ ID NO:41), and cloned into the BamHI/XbaIsites of pJAM2, yielding pJAM4. The gene encoding the M. tuberculosisDES antigen was amplified by primers JD17:(GGGTCTAGAACGACGGCTCATCGCCAGTTTGCC) (SEQ ID NO:42), and JD18:(CCCGGATCCATGTCAGCCAAGCTGACCGACCTG) (SEQ ID NO.:43) and also cloned intothe BamHI/XbaI sites of pJAM2 to give plasmid pJAM21.Expression and Purification of Recombinant Histidine-tagged Protein fromM. smegmatis

Plasmids pJAM4 and pJAM21 were introduced into M. smegmatis mc²155 andkanamycin resistant colonies grown in M63 medium [7.6×10⁻²M (NH4)₂SO₄,0.5M KH₂PO₄, 5.8×10⁻⁶M FeSO₄.7H₂O, pH 7] supplemented with 2% succinate(Sigma Chemical Co., St Louis, Mo.) for uninduced cultures or 2%succinate and acetamide (Sigma) for induced cultures. Bacteria weregrown for 3 days, after which cells were harvested and sonicated 4 timesfor 1 minute. Sonicates were analyzed for expression of recombinantproteins by SDS-PAGE and immunoblotting with the anti-35 kDa monoclonalantibody (mAb) CS38 for the M. leprae 35 kDa protein (CS38 supplied byProfessor Patrick Brennan, Colorado State University, Colorado) or forthe M. tuberculosis DES antigen using an anti-DES murine-derivedpolyclonal antibody. For protein purification, the sonicates wereapplied to Ni-NTA resin (Qiagen Inc., Calif.) and bound protein waswashed consecutively with 5 mM, 20 mM and 40 mM imidazole (Sigma) insonication buffer (1×PBS, 5% glycerol, 0.5 M NaCl and 5 mM MgCl₂).Protein was eluted with 200 mM imidazole in sonication buffer anddialyzed against PBS. Nonhistidine-tagged M. leprae 35 kDa proteinderived from M. smegmatis and the E. coli 35 kDa 6-histidine fusionprotein were purified as described previously (Triccas et al., 1996).

Protein Capture ELISA

ELISA plates were coated with the murine anti-M. leprae 35 kDa mAb ML03(50 mg/ml; supplied by Professor J. Ivanyi, Hammersmith Hospital,London, UK) and mycobacterial sonicates were added at a concentrationrange of 0.1 mg/ml to 100 mg/ml. Plates were blocked with 3% bovineserum albumin (BSA), washed, and anti-rabbit 35 kDa protein polyclonalantibody (1:1000) added. Binding was visualized using alkalinephosphatase conjugated anti-rabbit IgG (Sigma) andn-nitro-phenyl-phosphate (NPP) (1 mg/ml). Protein amount was determinedby comparison with purified M. leprae 35 kDa protein concentrationstandards (Triccas et al., 1996).

Assessment of Protein Binding to Leprosy Sera by ELISA

Microtitre plates were coated with antigen (100 μg/ml to 100 mg/ml)overnight at room temperature. Plates were washed, blocked with 3% BSA,and pooled sera (diluted 1:100) added for 90 minutes at 37° C. Plateswere washed, and alkaline phosphatase conjugated anti-human IgG (Sigma)added for 60 minutes at 37° C. Binding was visualized by the addition ofn-nitro-phenyl-phosphate (1 mg/ml) and absorbance was measured at 405nm.

Construction of the pJAM2 Vector and Utilization for Over-expression ofthe Gene Encoding the 35 kDa Antigen of M. leprae in M. smegmatis

The promoter region of the gene encoding the acetamidase of M. smegmatisNCTC 9449 permits the inducible expression of the enzyme in the presenceof the substrate acetamide (Mahenthiralingam et al., 1993). In order todetermine if the promoter could regulate the expression of foreign genesplaced under its control, the vector pJAM2 was constructed (FIG. 2A).This plasmid contains 1.5 kb upstream of the acetamidase coding region,DNA encoding the first 6 amino acids of the acetamidase gene, threerestriction enzymes sites, and the coding region for 6 histidineresidues. Thus this vector should allow for the inducible expression offoreign genes cloned within it, while also permitting simplepurification of the recombinant protein by virtue of the polyhistidinetag. In order to validate the system, the coding region of the M. leprae35 kDa protein was amplified and cloned into the BamHI/XbaI sites ofpJAM2 to give plasmid pJAM4. This protein is a major antigen of M.leprae and represents a promising candidate as a leprosy-specificdiagnostic reagent (Triccas et al., 1996). Plasmid pJAM4 was introducedinto M. smegmatis mc²155, and recombinant colonies grown in minimalmedia containing 2% succinate in the presence or absence of 2%acetamide. Sonicates were prepared and proteins analyzed by SDS-PAGE. Asshown in FIG. 10A, a prominent band was visible at around 37 kDa incells grown in acetamide plus succinate (lane 2), but absent from cellsgrown in succinate alone (lane 1). This band reacted in immunoblottingwith mAb CS38, which is raised against the native M. leprae 35 kDaprotein (FIG. 10B, lane 2)

Quantifying Expression of Recombinant Protein in M. smegmatis Using thepJAM2 Vector

In order to quantify the level at which the 35 kDa protein was beingproduced by virtue of the acetamidase promoter in M. smegmatis/pJAM4,antigen-capture ELISA was employed. As shown in FIG. 11, no protein wasdetected in M. smegmatis/pJAM4 grown in succinate alone. When the samestrain was grown in the presence of acetamide, the 35 kDa proteinrepresented approximately 8.6% of the total bacterial sonicate. Thestrength of expression was highlighted through comparison with proteinlevels in M. smegmatis harboring plasmid pWL19 (Winteret al., 1995),where expression of the 35 kDa protein-gene is driven by the β-lactamasepromoter of Mycobacterium fortuitum, one of the strongest mycobacterialpromoters characterized to date (Timm et al., 1994; Timm et al., 1994b).While M. smegmatis/pWL19 produced high levels of 35 kDa protein,representing 7.1% of the bacterial sonicate, this was around 17% lessrecombinant protein than that detected in M. smegmatis/pJAM4.

Purification of Histidine-tagged Protein from Recombinant M. smegmatis

We next determined if the high-level expression by virtue of the M.smegmatis acetamidase promoter could allow efficient purification of the35 kDa protein using the 6 histidine residues attached to itsC-terminus. This system has been successfully used in a number ofeucaryotic and procaryotic expression systems, and is favored due itssimple and reliable purification procedure, coupled with minimal effectsof the histidine tag on the target protein conformation, function, andimmunogenicity (Crowe et al., 1994). Although this system had not beenused in mycobacteria before, it seemed an ideal choice to allow thesimple and rapid purification of structurally and immunologically intactrecombinant mycobacterial proteins. Sonicates of M. smegmatis/pJAM4grown in the presence of acetamide were added to Ni-NTA resin (QiagenInc., Calif.), the column washed consecutively with varying amounts ofimidazole (5 mM, 20 mM and 40 mM) and protein eluted with 200 mMimidazole. This single-step procedure allowed 35 kDa protein ofpredominantly a single species to be purified (FIG. 11A, lane 3). Thepurified product reacted with the anti-M. leprae 35 kDa protein mAb CS38(FIG. 10, lane 3). Therefore the strategy of Ni-NTA affinitychromatography by virtue of a polyhistidine tag can be utilized for theefficient purification of recombinant proteins from mycobacteria.

Analysis of the Effect of the Histidine Tag on Recombinant ProteinConformation and Immunogenicity

Previously it was demonstrated that recombinant forms of the M. leprae35 kDa protein will only react with sera from leprosy patients if theprotein is produced in a conformation that resembles that of the nativeantigen (Triccas et al., 1996). This property allowed us to test theeffect, if any, of the histidine tag on the conformation of therecombinant 35 kDa protein. Three preparations of recombinant 35 kDaprotein were used: the histidine-tagged version purified in this study,a nonhistidine-tagged version purified from M. smegmatis, and an E. coli35 kDa 6-histidine fusion protein. The two latter proteins were purifiedas described previously (Triccas et al., 1996). The binding of pooledlepromatous leprosy sera to these three forms of the 35 kDa protein wereassessed by ELISA. The sera did not react with the E. coli form of the35 kDa protein (FIG. 12). By contrast, the 35 kDa-histidine fusionprotein purified from M. smegmatis/pJAM4 was strongly recognized by thesera. Furthermore, similar reactivity was exhibited towards the sameprotein purified from M. smegmatis containing no additional histidineresidues, suggesting that the addition of the histidine tag had noapparent effect on the conformation and indeed immunogenicity of therecombinant protein.

Induction and Over-expression of the Gene Encoding the M. TuberculosisDES Antigen Using the pJAM2 Expression System

To demonstrate that pJAM2 can be used for the induction and expressionof other genes placed within it, we cloned the gene encoding the M.tuberculosis DES antigen into the BamHI/XbaI sites of the vector, togive pJAM21. The DES antigen is an immunodominant B-cell antigen withsignificant sequence similarity to plant acyl-acyl carrier proteindesaturases (Jackson et al, 1997). As assessed by immunoblot, noexpression of the DES gene was observed in M. smegmatis alone grown inthe presence or absence of acetamide (FIG. 13, lanes 1 and 2), or by M.smegmatis harboring pJAM21 (strain MYC1553) grown in the absence ofacetamide (FIG. 13, lanes 3 and 5). By contrast, the DES antigen wasreadily detected in sonicates of MYC1553 grown in the presence of 2%acetamide (FIG. 13, lanes 4 and 6). These results indicate thathigh-level induction of the des gene could be achieved by use of thepJAM2 expression system.

The references cited herein are listed on the following pages and areexpressly incorporated by reference.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

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1. A process for producing a recombinant protein in a Mycobacteriumhost, comprising: a) cloning a DNA sequence comprising all or part of agene encoding a Mycobacterium protein into plasmid pJAM2, b) introducingthe recombinant pJAM2 obtained in a) into a Mycobacterium host, c)selecting transformed colonies of the Mycobacterium host, and d)cultivating the transformed colonies in a medium comprising acetamide toallow expression of the recombinant protein; wherein the recombinantprotein comprises a 6×HIS tag.
 2. The process of claim 1, wherein thecultivating comprises cultivating in a medium supplemented withsuccinate and acetamide.
 3. The process of claim 1, further comprisinglysing the cultivated cells to obtain the recombinant protein.
 4. Theprocess of claim 3, further comprising purifying the expressedrecombinant protein by immunoaffinity.
 5. The process of claim 1,wherein the Mycobacterium host is a Mycobacterium smegmatis strain. 6.The process of claim 1, wherein the all or part of a gene encoding aMycobacterium protein is obtained by DNA amplification of the gene. 7.The process of claim 1, wherein the recombinant pJAM2 is pJAM21 plasmidor pJAM4 plasmid.
 8. A process for producing a recombinant protein in aMycobacterium host, comprising: a) introducing recombinant plasmid pJAM2into a Mycobacterium host, wherein the recombinant plasmid pJAM2comprises a cloned DNA sequence comprising all or part of a geneencoding a Mycobacterium protein; b) selecting transformed colonies ofthe Mycobacterium host containing the recombinant plasmid pJAM2; and c)cultivating the colonies in a medium comprising acetamide to allowexpression of the protein; wherein the recombinant protein comprises a6×HIS tag.
 9. The process of claim 8, wherein the cultivating comprisescultivating in a medium supplemented with succinate and acetamide. 10.The process of claim 8, further comprising lysing cells of thecultivated colonies to obtain the protein.
 11. The process of claim 10,further comprising purifying the expressed protein by immunoaffinity.12. The process of claim 8, wherein the Mycobacterium host is aMycobacterium smegmatis strain.
 13. The process of claim 8, wherein theall or part of the gene encoding a Mycobacterium protein is obtained byDNA amplification of the gene.
 14. The process of claim 8, wherein therecombinant plasmid pJAM2 is plasmid pJAM21.
 15. The process of claim 8,wherein the recombinant plasmid pJAM2 is plasmid pJAM4.
 16. The processof claim 8, wherein the gene encoding the Mycobacterium protein isforeign to the Mycobacterium host.